1) Dilute cells 1:1 with trypan blue solution
a. Use separate eppendorf tube
b. 100 µL cells, 100 µL trypan blue is a good volume
2) Mix well and allow dye to permeate dead cells (~ 5 min)
3) Place cover on hemacytometer
4) Pipette cell/dye mixture into side channel until one side under cover is filled
5) Count only living cells (not blue) in large central block
6) Multiply by 2 to account for dilution
7) Multiply by 104 to obtain cells/mL
8) Repeat with other side of hemacytometer for average
9) Clean hemacytometer and cover by wiping with ethanol on a Kimwipe
Notes:
1) Total # cells/mL of original cell suspension = (# of cells in 4 boxes/4)x10,000xdf
2) Cell viability = # alive cells/ (# alive cells + # dead cells)